Immunotherapy in many different forms is making an increasingly strong impact on the treatment success in lymphoid malignancies. Monoclonal antibodies are a mainstay of treatment. In addition some novel agents, such as the immunomodulatory drugs thalidomide and lenalidomide appear to act primarily through an immune effect. In collaboration with the NCI lymphoma team, we have studied the effect of the monoclonal anti-CD20 antibody rituximab on tumor cells in patients with chronic lymphocytic leukemia (CLL). First, we identified about 80 genes up-regulated in tumor cells in response to rituximab infusion, many of which are known to be regulated by interferon (IFN) and to have pro-apoptotic function. We also found that IFN gamma was consistently upregulated in the serum within the first 6 hours of treatment indicating activation of immune effector cells. A surprising observation was that all these changes completely subsided by 24 hours. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used Western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2 hours from the start of rituximab resulting in virtually complete loss of expression at 24 hours. Next, we used ImageStream technology to directly visualize tumor-immune cell interactions in-vivo. We found transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. CD20 shaving appears to be a mechanism how tumor cells can resist treatment given that that persistent disease in the bone marrow was mostly CD20 negative. In parallel we also investigated changes in the function of NK cells and monocytes: consistent with NK cell activation we found an increase in CD69 after RTX administration and down-regulation of perforin expression. Activation of NK cells is triggered by the engagement of CD16/FcRIIIa by rituximab coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost and was not completely recovered by 24 hours. In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: both NK cells and monocytes isolated from patients during treatment showed a significant decrease in their ability to kill tumor cells. Collectively, our results identify loss of CD20 from CLL cells and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. We have recently initiated a new treatment trial for CLL patients using ofatumumab, a 2nd generation anti CD20 antibody. Preliminary results demonstrate that ofatumumab is stronger in activating complement mediated killing of CLL cells than rituximab, but it appears to also cause loss of CD20 on tumor cells. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. These mechanisms may be of general importance to immunotherapy of hematologic malignancies. Early gene expression studies revealed several genes that are expressed in CLL cells but not in normal B-cells. Such molecules could be valuable novel targets for immunotherapy. In collaboration with the Rader lab in NCI we focused on two such molecules: ROR-1 and TOSO. ROR-1 could be validated as a CLL specific antigen and efforts to use it as a target for immunotherapy are being pursued by the Rader lab. Work from other groups revealed that TOSO functions as the long sought Fc-receptor for IgM. TOSO is a transmembrane protein that is expressed on some normal B-cells but is highly overexpressed on CLL cells. We discovered that TOSO can rapidly uptake IgM, internalize it in specific vesicles, and transport it through the endocytic pathway to the lysosomes. Interestingly, aggregation of TOSO with IgM lead to rapid internalization of IgM whereas mAb bound TOSO was not internalized. Because of its restricted expression on CLL cells and its intracellular trafficking, we think that TOSO represents a potential means of delivering a cytotoxic agent into malignant cells. To this end, we engineered a protein backbone derived from IgM that can be conjugated to cytotoxic drugs. Preliminary data show that such constructs can selectively kill TOSO expressing cells and we are moving towards preclinical studies that should set the stage for possible clinical exploration of this approach as a treatment for CLL. Lenalidomide is an immune modulatory drug showing promising phase II results. Its mechanism of action in CLL is not well understood. In vitro data suggest that anti-leukemic immune responses are important. In vivo data that mechanistically link immune stimulation to clinical responses are lacking. We designed an independent, single center, phase II trial of lenalidomide in relapsed/ refractory CLL to address these questions. Of 32 patients enrolled on the trial 5 patients, achieved a response. WE obtained blood and lymph node samples for correlative analyses and performed flow cytometry, gene expression profiling and cytokine measurements. Key observations from these studies are that T cell numbers in the lymph node increased 1.4 fold during treatment in responding patient whereas there was no change in non-responding patients. Next we performed gene expression profiling on purified CLL cells and LN core biopsies obtained on day 8 of treatment and found that lenalidomide induced upregulation of 95 genes, many of which are known to be regulated by interferon gamma (IFNy). These data provide a first mechanistic link between the degree of lenalidomide induced immune activation and clinical response in CLL.